EP1981846A2

EP1981846A2 - Markers, method for manufacturing them and their applications

Info

Publication number: EP1981846A2
Application number: EP06794328A
Authority: EP
Inventors: François SCHERNINSKI; Vincent Guyon; Marie-Noëlle RAGER
Original assignee: Laboratoires Synth-Innove
Current assignee: Laboratoires Synth-Innove
Priority date: 2005-08-11
Filing date: 2006-08-11
Publication date: 2008-10-22
Anticipated expiration: 2026-08-11
Also published as: WO2007017602A3; US20120045851A1; DK1981846T3; US20080318336A1; FR2889700B1; US8034626B2; WO2007017602A2; PL1981846T3; CA2618217A1; FR2889700A1; PT1981846T; EP1981846B1; ES2657667T3; CA2618217C

Abstract

La présente invention porte sur les marqueurs susceptibles de former une liaison covalente ou non covalente avec une molécule cible, consistent en un colorant auquel est lié de fa?on covalente par un ou plusieurs carbones de sa structure chimique : - un ou plusieurs groupe(s) [FONC], et - eventuellement un ou plusieurs groupe(s) [SOL] ledit marqueur présentant la formule générale : (I) [COLOR] représentant Ie colorant ; [FONC] représentant chacun indépendamment un groupe -X-A-Z, dans lequel : - X est choisi dans le groupe consistant en un atome d'oxygène, un atome de soufre, un groupe NR1R2, R1 et R2 étant chacun indépendamment 1 ' un de l'autre un atome d'hydrogène ou un groupe alkyle, linéaire ou ramifié en C1-C30, de préférence en C1-C18, plus préférentiellement en C1-C5; - A est choisi dans le groupe consistant en un groupe alkylène ou un groupe alkylène-arylène ; - Z est une fonction chimique réactive ; [SOL] représentant chacun indépendamment un groupe -X' -A' -Z', dans lequel : - X' est choisi dans Ie groupe consistant en un atome d'oxygène, un atome de soufre, un groupe NR1R2, R1 et R2 étant chacun indépendamment l'un de 1 ' autre un atome d'hydrogène ou un groupe alkyle en C1-C30, de préférence en C1-C18, plus préférentiellement en C1-C5 ; - A' est choisi dans le groupe consistant en un groupe alkylène ou un groupe alkylène-arylène; - Z' est un groupement polaire ou apolaire.

Description

MARKERS, THEIR MANUFACTURING PROCESS AND THEIR

APPLICATIONS

The present invention relates to new markers that can be used for labeling target molecules. The invention also relates to a method for obtaining these markers. Finally, it concerns the application of these new markers for the detection and / or quantification of target molecules. The labeling of a target molecule and in particular of biological molecules by markers is of great interest in the field of cellular and molecular biology, in particular in cytology / histology, flow cytometry, for the sequencing of nucleic acids. In the field of medical imaging, the use of fluorescent markers makes it possible, among other things, to locate lesions or tumors.

Fluorescent markers are widely used in biology, biophysics, or physiology not only to study biological processes at the cellular and molecular level or quantify physiological substances to be analyzed but also in medicine in non-invasive imaging techniques for surveillance or diagnosis . Some specific examples of application of fluorescent markers in biology are:

- identification and separation of sub-population of cells in cell mixtures by flow cytometry or microscopy techniques, - determination of the concentration of a substance that binds to a second species such as an antibody antigen in immunoassay techniques, nucleic acid sequencing, nucleotide sequence matching, - the location of substances such as I ¹ DNA, proteins in electrophoresis gels and other insoluble supports by fluorescent staining techniques. In many techniques, including fluorescence optical microscopy, it is possible to simultaneously use several fluorescent markers capable of emitting at different wavelengths. This "multi-marking" is performed without any confusion between the signals occurring during the detection.

The principle of multi-labeling is also used in the particular technique of FRET (Fluorescence Resonance Energy Transfer) which exploits the energy transfer from the excited state of a donor fluorescent marker to a second fluorescent acceptor marker, this transfer of energy is only possible when both markers are nearby. This technique makes it possible to measure the interactions (association or dissociation) between two proteins, one of which is coupled to a donor fluorescent marker and the other is coupled to an acceptor fluorescent marker.

However, during these multi-markings, it is necessary to take into account the problems of possible partial overlaps of the emission spectra of the markers used which distort the reading of the results. Therefore, during multi-labeling, it is essential to limit the phenomenon of spectral overlap, which requires access to a wide range of markers whose spectral characteristics are completely controlled. The first object of the present invention consists of new markers composed of "functionalized dyes" whose resonance system is not affected by functionalization.

By "dye" is meant any colored substance, natural, artificial or synthetic, absorbing light in the field of ultraviolet, visible and / or infrared. In the context of the invention, the dyes may be fluorescent. "Fluorescent dyes" means dyes having the ability to be transiently excited by absorption of a light radiation and then to return to their initial state by emitting a radiation whose wavelength is higher than that of excitation radiation. By "functionalized dyes" are meant dyes having at least one reactive chemical function allowing their coupling to target molecules and possibly at least one function enabling them to be soluble under conditions of use. Markers of the prior art can be classified into three categories depending on how their reactive chemical function is related to the cyclic structure of the dye.

The markers of the first category are defined by the fact that their reactive chemical function is directly connected to a carbon atom of a ring of the hydrocarbon skeleton of the dye. They have been described in particular in ϋS5,627,027, US2002 / 077487, FR2764605, US4,614,723, JP03133629, JP03133628, JP63277680, Masiero, S. et al. "G-quartets as a self-assembled scaffold gold circular porphyrin arrays" Chemical Communications, vol.15, 2003 pages 1995-1996, Yu, Junhua et al. "Porphyrin capped TiO ₂ nanoclusters, tyrosine methyl ester enhanced electron transfer", FR2 748 027. However, the major disadvantage of such labels is that the reactive chemical function is located in close proximity to one cycle of the dye molecule . This proximity leads to a large steric hindrance which greatly hinders the coupling to target molecules. On the other hand, this proximity affects the absorption and fluorescence spectra of the dye. However, the choice of a dye for the synthesis of a marker is dictated by its spectral properties. Therefore, obtaining a marker whose coupling ability is hampered by a steric hindrance and whose spectral properties are different from those of the dye chosen is not particularly interesting.

In order to overcome the disadvantages mentioned above, a second category of markers has been developed.

The markers of this second category are defined by the fact that their reactive chemical function is indirectly linked via an alkyl chain to an atom which belongs to the molecule of the initial dye, this atom not being a carbon atom. . It is frequently a nitrogen atom, which most often belongs to the cyclic structure of this dye.

The manufacture of these markers of the second category is limited to the use of dyes consisting most often of one or more nitrogenous heterocycles. Each nitrogen atom of the initial dye carries a chain which strongly influences the solubility of the dye: it is often sulphoalkyl chains if the dye must be solubilized in an aqueous medium or alkyl chains if the dye must be solubilized in a hydrophobic medium. The technique for producing the markers of this second category consists in replacing one or more of these alkyl or sulphoalkyl chains by a hydrocarbon chain carrying the reactive chemical function. Nevertheless, this technique has the effect of significantly reducing the solubility of the marker relative to the initial characteristics of the dye. In addition, the manufacture of markers in this second category only gives access to a very limited number of entities because it is only applicable to a number of restricted dyes that contain one or more heteroatoms.

For example, the patents WO2005 / 058370, WO2003 / 082988, EP1209205 US 6,027,709 and US 6,224,644 disclose markers of this second category.

No. 6,027,709 describes carbocyanine dyes in which a sulphoalkyl chain has been replaced by an alkyl chain carrying a carboxylic acid function (electrophilic reactive chemical function).

US Patent 6,224,644 also discloses similarly modified carbocyanines for use as biomolecule markers. However, these markers are distinguished from those of US Pat. No. 6,027,709 in that the reactive chemical function is not electrophilic but nucleophilic (hydroxyl, amine and thiol function).

This class of markers is therefore of limited interest since it only applies to a few carbocyanine-type dyes with a reduction in the solubility of the label relative to that of the initial dye, whereas it would have been desirable to increase this solubility to facilitate coupling with a large number of target molecules. In the case where the water solubility of the label is desired, the direct addition of groups such as NO ₂ and SO ₃ ^" on the aromatic rings alleviates this problem but affects the absorption and fluorescence spectra of the initial dye.

A third class of markers, as described in EP 1 209 205, WO02 / 32466, WO94 / 08631 and Lobnik, A. et al. "PH optical sensors based on sol-gels. Chemical Doping versus Covalent Immobilization "Analytica Chimica Acta, vol.367, no.1-3, 1998, pages 159-165, is defined by the fact that their reactive chemical function is connected indirectly via an alkyl or amidoalkyl chain to a carbon atom, and not a nitrogen atom, of the cyclic structure of the dye, thereby avoiding the deletion of any of the sulfoalkyl or alkyl chains which condition the solubility of the marker.

Nevertheless, the manufacture of the markers of this third category gives access to a number of entities even more limited than those of the second category because they have the major disadvantage of being difficult to synthesize. Indeed, the precursors for synthesizing such markers are not currently commercially available and their synthesis requires the implementation of many steps with low final yields.

To date, the synthesis of functionalized dyes serving as markers for detecting and or quantifying target molecules is known. However, the markers of the prior art have at least one of the following three basic drawbacks, which considerably limit their use: the reactive chemical function of these markers is in the immediate vicinity of a dye cycle, which results in a Steric hindrance and strongly hinders the coupling to target molecules, thus rendering them useless. Moreover, their spectral properties are different from those of the dyes from which they are obtained, because of the electronic effects that occur between the reactive chemical function and the aromatic rings of the dye.

These markers are hardly soluble in the coupling medium whose nature is dictated by the identity of the target molecule.

These markers can not be synthesized from reagents that are easily accessible commercially and according to simple and rapid synthesis methods; of this makes the available number of these markers is limited.

The present invention proposes in turn to prepare according to a particular method markers of a fourth category having none of the disadvantages of the markers of the prior art.

Furthermore, to the knowledge of the applicant, there has never been described in the prior art a method for easily functionalizing a very wide panel of dyes. Indeed, the methods of the prior art can only be applied to a small number of dyes while there are a large number of dyes with specific spectral characteristics of interest for the labeling of target molecules. In addition, during multi-tagging, it is essential to take into account possible partial overlap problems of the emission spectra of the markers used which distort the reading of the results. In the context of the present invention, this problem of overlap is minimized insofar as the user has a large panel of markers, possibly fluorescent having different spectral properties, that is to say wavelengths. of different excitation and emission over a wide region of the detection spectrum.

The advantage of the method of the invention is that it makes it possible to select a dye according to its particular spectral characteristics and to be able to easily functionalize it without significantly altering its spectral characteristics or its solubility characteristics.

In view of the prior art, it is apparent that there is a need for a wide variety of easily synthesized markers from dyes whose choice is mainly dictated by their absorption and emission properties, said markers having not only substantially the same spectral properties as the dyes from which they are obtained but also a solubility in the coupling medium which is equivalent or even improved with respect to the dye initial.

The inventors, after extensive research, had the merit of finding that it was possible to obtain a large panel of new markers combining all the functional and spectral characteristics sought from a large panel of dyes or dyes. intermediates commonly available commercially.

The first subject of the present invention relates to novel markers capable of forming a covalent or non-covalent bond with a target molecule, consisting of a dye to which one or more carbons of its chemical structure is covalently bound: one or more groups ( s), [FONC], and optionally one or more group (s) [SOL], said marker having the general formula:

[COLOR] representing a molecule selected from the group consisting of phtaiines, carbocyanines, merocyanines, porphyrins, phthalocyanines; [FONC] each independently representing a group -XAZ, in which:

X is selected from the group consisting of oxygen, sulfur, NR ₁ R ₂ , R 1 and R ₂ are each independently hydrogen or alkyl , linear or branched C ₁ -C ₃₀ , preferably C ₁ -C ₁₈ and more preferably C ₁ -C _5;

A is selected from the group consisting of an alkylene group or an alkylene-arylene group; Z is a reactive chemical function;

[SOL] each independently representing a group -X '-A' -Z ', wherein:

X 'is selected from the group consisting of an oxygen atom, a sulfur atom, a group NR ₁ R ₂ , R _{1 and} R ₂ each independently of one another a hydrogen atom or an alkyl group linear or branched C ₁ -C ₃₀ , preferably C ₁ -C ₁₈ and more preferably C ₁ -C _5;

A 'is selected from the group consisting of an alkylene group, or an alkylene-arylene group;

Z 'is a polar or apolar group.

In the present invention, "phthalein" means dyestuffs having the following structure (1):

[COLOR] =

(1) "carbocyanines" means dyestuffs having the following structure (2): [COLOR] =

(2) "Merocyanines" means dyestuffs having the following structure (3):

: 3)

"porphyrins" means the dyestuffs having the following structure (4):

[COLOR] =

(4) "phthalocyanines" means dyestuffs having the following structure (5):

[COLOR] =

(5)

in which :

- Mi to M ₄ , Mg to Mi ₂ , Qi to Qi ₂ , Di to D ₁₆ , which are identical or different from each other, are chosen from the group comprising the following radicals or groups: hydrogen, hydroxyl, halogen, acetyl amine, substituted amine, quaternary ammonium, phosphate, nitro, carboxylic acid and its salts, sulfonic acid and its salts, alkylcarboxy of 2 to 30 carbon atoms, alkyl, linear or branched, having 1 to 30 carbon atoms, cycloalkyl having 3 to 14 carbon atoms, alkyloxy having 1 to 30 carbon atoms, haloalkyl having 1 to 30 carbon atoms, hydroxyalkyl having 1 to 30 carbon atoms, alkyl ester having 2 to 40 carbon atoms, nitroalkyl having 1 to 30 carbon atoms, carboxyalkyl having 2 to 30 carbon atoms, aminoalkyl having 1 to 30 carbon atoms, sulfoalkyl having 1 to 30 carbon atoms, aryl, aryloxy, arylalkyl, haloaryl, aryl ester, Ch / Qe _r Q9 and Q ₂ may not represent aryl or aryloxy, with the proviso that at least one of Qi to Q ₂ represents H or an aromatic ring, provided that at least one of Di Die represents H;

Z ₁ and Z ₂ are each independently of one another the atoms necessary to complete an indole, benzindole or naphthindole ring; Z ₃ represents O or S;

V and W are each independently of one another selected from CR ₇ R ₈ , O, S, Se and NR 9, wherein R ₇ , R ₉ and R ₉ are each independently selected from hydrogen and group (CH ₂ ) _m Ri _{0 /} or m is an integer from 1 to 18 and R ₀ is selected from hydrogen, amine, substituted amine, quaternary ammonium, aldehyde, halogen, cyano, aryl, heteroaryl, hydroxyl, amide, acid sulfonic acid and its salts, carboxylic acid and its salts; n is an integer from 1 to 10, preferably from 1 to 7 and more preferably from 1 to 3; i is an integer from 1 to n;

R ₃ to Re represent, independently of one another, a hydrogen atom, a linear or branched C ₁ -C 30, preferably C ₁ -C 18, more preferably C ₁ -C ₅ , cycloalkyl, aryl or aryloxy alkyl group; nitroalkyl, alkylamine, substituted alkylamine, alkylammonium quaternary, alkylphosphate, alkylsulfonic acid and its salts; - Ti to Ts, each independently represent a hydrogen atom, a halogen atom, a linear or branched C ₁ -C ₃₀ , preferably C ₁ -C 18, more preferably C ₁ -C ₆ alkyl group; ₅ , sulfoalkyl, cycloalkyl, aryl, aryloxy, nitro, amine, substituted amine, quaternary ammonium, phosphate, sulfonic acid and its salts, selected from hydrogen and an alkyl group more preferentially in with Ru as defined previously; are each independently of one another a hydrogen atom, a linear or branched alkyl group preferably more preferably in cycloalkyl, aryl;

Bu, B2i, B ₃ each independently represent a cycloalkyl methine group monounsaturated or diunsaturated by 4 to 8 carbon atoms, mono- or diunsaturated aryl-cycloalkyl of 4 to 8 carbon atoms, or one of the following groups:

each of these groups being either unsubstituted or substituted by one or more of the following groups: alkyl linear or branched Ci-Cis, preferably C ₅ -C, halogen, sulfonate, sulfoalkyl, aryl, sulfoaryl, aryloxy, hydroxy, hydroxylate, ketone, nitro, amino, substituted amino, quaternary ammonium; Y represents a counterion chosen from the following ions: halide, p-toluenesulphonate, methanesulphonate, trifluoromethanesulfonate, perchlorate, acetate, sodium, potassium, calcium, magnesium, lithium, ammonium and trialkylammonium; - P is an integer from 0 to 8 necessary for the neutrality of the molecule.

The term "reactive chemical function" denotes any functional group capable of binding by covalent or non-covalent bond (electrostatic, hydrogen, coordinative, ionic or complex) directly or after activation, with at least one of the functions naturally occurring. present or artificially introduced on a target molecule. By way of nonlimiting examples of suitable reactive chemical functions for the purposes of the invention, mention may be made in particular of the carboxylic acid and its salts functions, sulphonic acid and its salts, acid anhydride, acid chloride, ester (alkyl ester, p-nitrophenyl ester, succinimidyl ester, sulfosuccinimidyl ester, etc.), azido (acyl azide, azidonitrophenyl, etc.), hydrazide, 3-acyl-1,3-thiazolidine-2-thione, amine, amine substituted, quaternary ammonium, isocyanate, isothiocyanate, hydrazine, phthalimido, maleimide, haloacetamide, monochlorotriazine, dichlorotriazine, mono or dihalogenated pyridine, mono or dihalogenated diazine, aziridine, thiol, sulfonyl chloride, vinylsulfone, disulfide, methanethiosulfonate, hydroxyl, phosphoramidite, epoxy , aldehyde, carbonate, glyoxal, imidazolyl. The expression "polar or apolar group" denotes any functional group capable of facilitating the solubilization of a chemical compound in polar or apolar solvents. By way of nonlimiting examples of polar groups suitable for the purposes of the invention, mention may be made in particular of the groups, carboxylic acid and its salts, sulphonic acid and its salts, amine, substituted amine, quaternary ammonium, carbohydrate, glycol, hydroxyl , nitro, phosphate. By way of nonlimiting examples of nonpolar groups, mention may be made especially of linear or branched alkyl groups having from 1 to 30 carbon atoms, aryl, cycloalkyl having from 3 to 14 carbon atoms and alkyloxy having from 1 to 30 carbon atoms. , haloalkyl having 1 to 30 carbon atoms, hydroxyalkyl having 1 to 30 carbon atoms, alkyl ester having 2 to 40 carbon atoms, aryloxy, arylalkyl, substituted arylalkyl, haloaryl.

By "alkylene" is meant a hydrocarbon chain, cyclic, linear or branched, having two free bonds, containing from 1 to 30 carbon atoms, preferably from 1 to 18 carbon atoms and more preferably still from 1 to 5 carbon atoms. carbon and can be in particular a methylene, ethylene, propylene, 2-methylpropylene, n-butylene, i-pentylene, n-pentylene, hexylene, heptylene, octylene, nonylene, or a cyclopentadiene group.

"Arylene" denotes an aromatic group having two free bonds, containing one or more aromatic rings, optionally substituted, in particular a phenylene which may be substituted or unsubstituted.

"Alkyl" and "C1-C30 alkyl" denotes a hydrocarbon chain, cyclic, linear or branched, having a free bond, containing from 1 to 30 carbon atoms, preferably from 1 to 18 carbon atoms and more preferably still from 1 to 5 carbon atoms and which can be in particular a methyl, ethyl, n-propyl, isopropyl or propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2,2-dimethylbutyl, 2-methylpentyl, 2,2-dimethylpropyl, isopentyl, neopentyl, 2-pentyl, hexyl, 2-hexyl, 3-hexyl, 3-methylpentyl, heptyl, octyl, nonyl, decyl, dodecyl. "Target molecule" denotes a biological or non-biological molecule intended to be coupled to a marker. This term includes but is not limited to organic molecules, polymers, silicate, natural or synthetic materials, lipid vesicles, amino acids, nucleic acids, nucleotides, oligonucleotides, peptides, proteins, carbohydrates, oligosaccharides, polysaccharides, antibodies, antigens, cell receptors, haptens, pectins, cytokines, hormones, toxins, bacteria, viruses, eukaryotic cells.

The marker according to the invention is characterized in that each of the [FONC] groups or [SOL] groups is attached to a carbon of the chemical structure of the dye via its respective group X or X 'such that previously defined. As a result, the hydrophilic or hydrophobic groups initially present on the dye and which may be carried by the nitrogen atoms or by any other atom of the structure, are not modified.

Specific but non-limiting examples of the [FONC] group are given below (Su represents the succinimidyl group): -X- (CH ₂ ) _r -C00Su, -X- (CH ₂ ) _r -COOSuSO ₃ Na, -X - (CH ₂ ) _r -COOH, -X- (CH ₂ ) _r -SO ₃ H, -X- (CH ₂ ) _E -SO ₃ Na, -X- (CH ₂ ) _r -COO-C ₆ H ₄ -NO ₂ , -X- (C ₆ H ₄₎ - (CH ₂₎ _E -C00Su, -X- (CH ₂₎ _E -NHCOCH ₂ I, -X- (CH ₂₎ _r -NCS, -X- (CH ₂₎ _r -C ₆ H ₄ CH (CH ₃ ) -COOSu, -N (CH ₃ ) ₂ - (CH ₂ ) _r -SO ₃ H, -X- (CH ₂ ) _r -OP [N (iPr) ₂ ] [CH ₂ CH ₂ CN] , X is as defined above, r is an integer from 1 to 18, preferably from 2 to 10, and more preferably from 3 to 5.

In general, the coupling reaction conditions between a label and a target molecule are dictated by the nature of the target molecule. In the case of labeling certain fragile biological molecules, in particular proteins, it is preferable that the coupling reaction be carried out in aqueous solution in order to avoid denaturation thereof. For this purpose, it is advantageous for the marker to have one or more Z 'groups conferring on it a water-soluble character in the non-denaturing coupling medium for the protein. On the other hand, for other target molecules, hydrophobic or nonpolar groups are preferable. In this case, the marker must preferably have at least one Z 'group conferring a hydrophobic or non-polar character.

These hydrophilic or lipophilic groups (Z ') may optionally be introduced in the same way as the reactive chemical function Z. Specific but nonlimiting examples of a group [SOL] comprising a group Z', hydrophilic or lipophilic, illustrating this structural analogy are given below:

-X- (CH ₂ ) _n -SO ₃ Na, -X- (CH ₂ ) _r -SO ₃ H, -X- (CH ₂ ) _r -C ₆ H ₃ (NO ₂ ) ₂ , -X- (CH ₂ ) ₂ ) _E -CH ₃ , X is as defined above, r is an integer from 1 to 18, preferably from 2 to 10, and more preferably from 3 to 5.

The markers of the invention may be fluorescent, when they are made from fluorescent dyes, and have fluorescent spectral characteristics ranging from ultraviolet to -X- (CH ₂ ) _r -SO ₃ H, -X- (CH ₂ ) _E -SO ₃ Na, -X- (CH ₂ ) _E -COO-C ₆ H ₄ -NO ₂ , -X- (C ₆ H ₄ ) - (CH ₂ ) _r -COOSu, -X- (CH ₂ ) _r -NHCOCH ₂ I, -X- (CH ₂ ) _r -NCS, -X- (CH ₂ ) _E -C ₆ H ₄ CH (CH ₃ ) -COOSu, -N (CH ₃ ) ₂ - (CH ₂ ) _r -SO ₃ H, -X- (CH ₂ ) _r -OR [N (iPr) ₂ ] [CH ₂ CH ₂ CN] , X is as defined above, r is an integer from 1 to 18, preferably from 2 to 10, and more preferably from 3 to 5.

-X- (CH ₂ ) _r -SO ₃ Na, -X- (CH ₂ ) _r -SO ₃ H, -X- (CH ₂ ) _r -C ₆ H ₃ (NO ₂ ) ₂ , -X- (CH ₂ ) ₂ ) _r -CH ₃ , X is as defined above, r is an integer from 1 to 18, preferably from 2 to 10, and more preferably from 3 to 5.

The markers of the invention may be fluorescent, when they are made from fluorescent dyes, and have fluorescent spectral characteristics ranging from ultraviolet to 1 'infrared.

Thus, the invention relates to markers derived from dyes chosen from the group comprising the following dyes: phthalein, carbocyanines, merocyanines, porphyrins, phthalocyanines said dyes being as defined above.

The label according to the invention obtained from a phthalein, is a compound having the structure (6),

wherein: each of M ₅ to M ₈ is as previously defined; each of M 'to M ₄ and M ₉ to M _12, independently of one another can have the definition given above for M ₁ to M _4, and M ₉ to Mi _2, or can represent [ SOL] or [FONC], [SOL] and [FONC] being as defined above, and provided that at least one of M'i to M ' ₄ and M' 9 to M '12 represents [FONC] .

According to a particular embodiment of the structure marker (6), at least one of M'i, M ' ₂ , M' ₃ or M ' ₄ represents [FONC], and / or at least one of M ' ₉ , M'i ₀ , M'u or M' ₁₂ represents [FONC]. The marker according to the carbocyanine type invention is a compound having the following structure (7)

(7) in which are as defined above, each of independently of each other, may have the definition given previously for or may represent [SOL] or [FONC], [SOL] and [FONC] being as previously defined, and provided that at least one of them represents [FONC].

According to a particular embodiment of the structure marker (7), at least one of and / or at least one among represents [FONC].

The marker according to the invention of merocyanine type is a compound having the following structure (8):

(8) in which

are as defined above, each of independently of one another, may have the definition given above for or may represent [SOL] or [FONC], [SOL] and [FONC] being as defined above, and provided that one at least from T 1 to T 1, represents [FONC].

The marker according to the invention of the porphyrin type is a compound having the following structure (9):

in which

Q _3, Q _6, Q ₉ and Q ₁₂ are as defined above, each of Q _'x Q' _2, Q _4, Q _5, Q _7, Q _8, Q ₁₀ and Q ' ₁₁ independently of one another may have the definition given previously for Q ₁ , Q ₂ , Q ₄ , Q ₅ , Q ₇ , Q ₈ / Q ₁₀ and Q _{1 1} or may represent [SOL] or [FONC] , [SOL] and [FONC] being as defined above, and provided that at least one of Q'i, Q ' ₂ , Q' ₄ , Q ' ₅ , Q' ₇ , Q ' ₈ , Q _'10 and Q ₁₁ represents [FUNC].

The marker according to the invention of phthalocyanine type is a compound having the following structure (10): 21

Io)

in which each of Di 'to Dis', independently of one another, can have the definition given previously for Di to Di ₆ , or can represent [FONC] or [SOL], [FONC] and [SOL] being as defined above, and provided that at least one of D'i to D'is represents- [FONC].

According to one embodiment of the invention, the marker is water-soluble, preferably at a concentration greater than 0.5% (w / v), more preferably at a concentration greater than or equal to 2% (w / v), and even more preferably still greater than 10% (m / v).

According to another embodiment of the invention, the marker is liposoluble, preferably at a concentration greater than 0.5% (w / v), more preferably at a concentration greater than or equal to 2% (w / v), and even more preferably still greater than 10% (m / v).

Specific examples of markers according to the invention are the following compounds: (1)

wherein each of y, z, which is the same or different, is an integer equal to 1, 2, 3, 4, 5, β, 7 or 8, Z represents -COOH or

Y represents SO ₃ - or SO ₃ Na

(2)

wherein each of y, z and n, which are the same or different, is an integer of 1, 2, 3, 4, 5, 6, 7 or 8, Z represents -COOH or

Y represents SO ₃ or SO ₃ Na

in particular

wherein each of y, z and n, identical or different, is an integer equal to 1, 2, 3, 4, 5, 6, 7 or 8

Z represents -COOH or

Y represents SO ₃ ^" or SO ₃ Na

in particular

(3)

wherein each of y, z and n, which are the same or different, is an integer of 1, 2, 3, 4, 5, 6, 1 or 8

Z represents -COOH or

Y represents SO ₃ or SO ₃ Na in particular

(5)

wherein each of y, z and n, which are the same or different, is an integer of 1, 2, 3, 4, 5, 6, 7 or 8

Z represents -COOH or

Y represents SO ₃ ^" or SO ₃ Na in particular

(6)

wherein each of y and z, identical or different, is an integer equal to 1, 2, 3, 4, 5, 6, 7 or 8, Z represents -COOH or

Y represents SO ₃ ^~ or SO ₃ Na in particular

(7)

Y represents SO ₃ - or SO ₃ Na in particular

The method for preparing the markers of the invention as described above, and in particular the introduction onto a carbon atom of the structure of the dye, of at least one [FONC] group, allowing said markers to be conjugated to a molecule target in a given coupling medium, and optionally the introduction of at least one group [SOL], constitutes the second object of the invention.

The method according to the invention by which it has been possible to synthesize markers according to the invention, is characterized in that it comprises a nucleophilic substitution reaction between: or: Z-A-L and [COLOR '] - Nu

or :

[COLOR ''] -L and Z-A-Nude

Z and A being as defined above; - L representing a leaving group;

Wherein N is a nucleophilic group selected from the group consisting of -OH, -SH or -NR ₁ R 2, R 1 and R 2 each independently of one another is a hydrogen atom or an alkyl group, linear or branched, C ₁ -C _30, preferably Cχ-Ci _8, most preferably Ci-C _5;

- [COLOR '] and [COLOR "] representing [COLOR] or a precursor or intermediate of synthesis of [COLOR],

[COLOR] being as defined previously. By "leaving group" is meant a group that can be replaced by another group in a substitution reaction.

Said leaving group may in particular be a halogen or a methanesulphonate group, para-toluenesulphonate or a diazonium group. This list is not exhaustive. The leaving group therefore allows the binding of the chain bearing either the reactive chemical function or the polar or apolar function on a carbon atom of the cyclic structure of the dye or its synthesis intermediate.

According to a particular embodiment, the nucleophilic substitution reaction can be carried out by opening a cyclic molecule of formula:

According to a particular embodiment, the nucleophilic substitution reaction may be a Williamson type reaction, carried out between the [COLOR '] -Nu molecule and the Z - A - L molecule, in the presence of a base.

Said base may especially be sodium hydroxide, potassium carbonate, potassium t-butoxide, sodium hydride or sodium amide. This list is not exhaustive.

The invention also relates to processes for the preparation of [COLOR '] -Nu and [COLOR "] -L.

[COLOR '] -Nu is obtained by the following successive reactions:

1. nitration of [COLOR '];

2. reduction of the product obtained in step 1;

3. optionally: either alkylation of the product obtained in step 2, or diazotization of the product obtained in step 2 to obtain a diazonium salt and nucleophilic substitution on this diazonium function. [COLOR ''] -L is obtained by the following successive reactions: the. nitration of [COLOR '']; 2 '. reduction of the product obtained in step 1; 3 '. diazotization of the product obtained in stage 2 'to obtain a diazonium salt;

4 '. optionally: nucleophilic substitution on the diazonium function of the product obtained in step 3 '.

According to a particular embodiment, [COLOR '] -Nu and [COLOR' '] -L represent the dye itself, that is to say [COLOR].

The nitration can be carried out for example by the action of nitric acid, nitronium tetrafluoroborate, sodium nitrate or sodium nitrite in an acidic medium. The reduction of the nitro derivative to amine can be carried out by catalytic hydrogenation, by hydrogen transfer in the presence of a catalyst or by the action of stannous chloride in acidic medium for example. This amino function can itself be used as a nucleophilic group or can be converted into another nucleophilic group. Since it is preferable to obtain another nucleophilic group, it can easily be obtained by substitution of the corresponding diazonium salt. In this case, the preceding amine function is converted into a diazonium salt by the action of sodium nitrite in an acidic medium. To obtain a hydroxy function, the diazonium salt is hydrolyzed in dilute sulfuric medium, for example. In order to obtain a thiol function, the diazonium salt is reacted with a sulfur compound, for example a xanthate, and then cleaved.

The moment in the synthesis where the introduction of the nucleophilic group Nu (or of the leaving group L) then the substitution reaction with the molecule ZAL (or ZA-Nu) will be carried out, that is to say the choice of the precursor [ COLOR '(or [COLOR'']), of course depends on the nature of the dye used. The person skilled in the art will of course be able to choose this moment so as to to limit the protection and deprotection operations of sensitive functional groups.

In the case where the synthesis of the functionalized dye must begin with one of its synthesis intermediates bearing the nucleophilic group Nu, it is recommended to protect this group beforehand in order to avoid unwanted side reactions.

Said nucleophilic group may be protected by a protective group, for example in the form of an ether (methoxy ...) for a hydroxyl group, a thioether

(benzylthio ...) for a thiol group, an amide or a urethane type group for example a t-butyloxycarbonyl group or a benzyloxycarbonyl group for an amine. After obtaining the dye or one of its synthetic intermediates, while the last synthesis steps are no longer likely to interfere with the reactive chemical function, the nucleophilic group is deprotected under the action of an acid, of a base or by reduction or oxidation and is then reacted with the ZAL molecule to introduce the reactive chemical function Z.

According to a particular embodiment, the method of the present invention may comprise the attachment of one or more [SOL] groups by using one or more of the chemical reaction steps previously defined for the fixation of the group.

[FONC]. In this case, the nucleophilic substitution reaction is carried out using a molecule Z '-A' -L (or Z '-A' -Nu) in which:

Z ', A', Nu and L are as defined above.

Thus, the method may comprise a nucleophilic substitution reaction between: Z '-A ¹ -L and [COLOR'] - Nude

or else: [COLOR ''] -L and Z '-A' -Nu -Z ¹ and A ⁽ , L, Nu, [COLOR '] and [COLOR "] being as previously defined;

- [COLOR '] - Nu and [COLOR "] -L being prepared according to the process described above.

The method of the invention thus makes it possible to obtain, from easily accessible dyes, new functionalized derivatives whose spectral characteristics and solubility characteristics are particularly interesting for the labeling of the target molecules.

Examples of reaction schemes for preparing markers from carbocyanines (Schemes Ia and Ib), phthalein (Scheme II) and porphyrins (Scheme III) are given below in a nonlimiting manner. In these reaction schemes, NHS is N-hydroxysuccinimide and DCC is dicyclohexylcarbodiimide.

(diagram Ia)

(diagram Ib)

(diagram II) The application of the markers of the invention, as described above, to the detection and / or quantification of target molecules constitutes the third subject of the invention. As indicated above, the markers of the invention are suitable as agents for labeling target molecules, the labeling being carried out by coupling of said marker by covalent or non-covalent binding with the target molecule to be assayed or detected. This coupling can be carried out according to conventional coupling methods used in this field.

According to a particular embodiment, the target molecule is reacted with 1 to 200 equivalents, more preferably 3 to 20 equivalents of the marker. When the target molecule is water-soluble, the reaction takes place in an aqueous buffer solution, preferably based on phosphate, carbonate or borate, whose pH is between 7.0 and 11.0. When the target molecule is fat-soluble, the reaction takes place in an organic solvent. The coupling reaction can also be carried out in the presence of a peptide coupling agent, for example carbodiimide reagents such as DCC (dicyclohexylcarbodiimide), carbonyldiimidazole, IDDQ (1-isobutyloxycarbonyl-2-isobutyloxy-l, 2- dihydroquinoline), phosphonium reagents such as BOP (benzotriazol-1-yloxytris- (dimethylamino) phosphonium hexafluorophosphate), uronium-type reagents such as HBTu (o-benzotriazolyl-tetramethyluronium hexafluorophosphate) and TBTu (o-benzotriazolyltetramethyluronium tetrafluoroborate) Woodward reagents such as N-ethyl-5-phenylisoxazolium-3'-sulfonate, Curtius reagents (hydrazine and nitrite).

The coupling reaction is carried out if necessary in presence of an organic base and dimethylsulfoxide, or preferably dimethylformamide.

The labeled target molecule is isolated from the reaction medium and purified by separation on silica gel, gel permeation and / or ultrafiltration.

The invention also relates to a method for detecting a biological or non-biological molecule comprising the coupling of a marker according to the invention with said molecule, and the actual detection of said molecule coupled to the marker by absorption spectrometry, fluorescence spectrometry, infrared spectrometry, electrophoresis, medical imaging in infra-red or near infra-red

(NIRS, Near Infra-Red Spectroscopy). This list is not exhaustive.

The invention also relates to precursors for synthesizing the markers of the invention, which are novel products consisting of molecules of general formula:

pY

in which

Z 1, V, p, Y are as defined above, R ' ₃ represents an electron pair or represents R 3 as defined above; μ is an integer equal to 0 or 1;

R ₁₂ represents an alkyl group, linear or branched, saturated or unsaturated C ₁ -C ₃₀ , preferably C ₁ -C ₈ , plus preferably C ₁ -C ₅ , sulphoalkyl, cycloalkyl, aryl, aryloxy, preferably a methyl group, each of T'i to T ' ₄ , independently of one another, may have the definition given above for Ti to T ₄ or may represent [SOL] or [FONC], [SOL] and [FONC] being as defined above, and provided that at least one of T ^r i to T \ is [FONC].

Nonlimiting examples of precursors are given below:

The invention will be described in more detail with the aid of the following examples which are not limiting but relate to advantageous embodiments. EXAMPLES

EXAMPLE 1

5-nitro-2,3,3-trimethyl- (3H) -indole (product A)

To a mixture of 6.4 g of 2,3,3-trimethyl- (3H) -indole and 50 ml of sulfuric acid cooled to 0-5 ^° C. is added, without exceeding 5 ° C., a solution of 3, 4 g of sodium nitrate in 100 ml of sulfuric acid. After stirring for ¹ hour at 0-5 ^° C., the reaction medium is diluted in 600 ml of water and neutralized by addition of solid sodium hydroxide. The precipitate formed is filtered and solubilized in 200 ml of ethyl acetate. After washing with water, drying over magnesium sulphate and evaporation, 8 g of product A are obtained (yield: 97.9%).

EXAMPLE 2

5-amino-2,3,3-trimethyl- (3H) -indole (product B)

A mixture of 6.6 g of product A, 43.7 g of stannous chloride dihydrate and 215 ml of hydrochloric acid is refluxed for two hours. After cooling to room temperature and filtration, the solid collected is solubilized in 130 ml of water. This solution is neutralized by adding 20% sodium hydroxide. The precipitate formed is filtered and then washed with water and dried under vacuum in the presence of phosphorus pentoxide. 5.4 g of product B (yield: 96%) are obtained. EXAMPLE 3

5-hydroxy-2,3,3-trimethyl- (3H) -indole (product C)

A mixture of 3.5 g of product B, 5 ml of sulfuric acid and 25 ml of water is cooled to 0 ^° C. A solution cooled to 0-5 ^° C. of 1.6 g of sodium nitrite in 4 ml of water is added without exceeding 5 ° C. After stirring at 0-5 ^° C. for 10 minutes, the reaction medium is slowly added to a mixture of 15 ml of sulfuric acid and 20 ml of water brought to 90 ^° C. After stirring at this temperature for one hour and then cooling at room temperature, the reaction medium is neutralized by addition of 20% sodium hydroxide. The precipitate formed is filtered and then washed with water and dried under vacuum in the presence of phosphorus pentoxide. 2.6 g of product C (yield: 74.3%) are obtained.

EXAMPLE 4

5-methoxy-2,3,3-trimethyl-benz (e) indole (product D)

A mixture of 35.4 g of 7-methoxy-2-naphthylhydrazine and 65 g of sodium hydrogen sulphate monohydrate in 180 ml of water is heated at 90 ^° C. for 15 min and then 23 g of 3-methyl-2-butanone. are added together. The reaction medium is maintained at 90 ^° C. for 7 hours, cooled to room temperature and then extracted with dichloromethane. The organic phase is washed with water and then evaporated under vacuum. The residue is purified by chromatography on silica gel (dichloromethane / methanol: 10 / 0.2). 28.9 g of product D are obtained (yield: 64.2%).

EXAMPLE 5

5-hydroxy-2,3,3-trimethyl-benz (e) indole (product E)

8.2 g of product D are introduced into a three-necked 250 ml. 82 ml of HBr (33% in acetic acid) are added. The mixture is maintained at 70 ^° C. for 7 hours and then cooled to ambient temperature. 2.5 liters of water and then 120 ml of 5M sodium hydroxide are added to the reaction medium. The precipitate formed is filtered and then washed with water and dried under vacuum in the presence of P ₂ O ₅ . 6.8 g of product E are obtained (yield: 88.7%).

EXAMPLE 6

5- [(5-carbethoxypentyl) oxy] -2,3,3-trimethylbenz (e) indole (product F)

A mixture of 20 g of product E, 21.7 g of ethyl 6-bromohexanoate and 13.5 g of potassium carbonate in 120 ml of acetone is refluxed for 8 hours and then cooled to room temperature. The reaction medium is filtered and evaporated under vacuum. The residue is dissolved in 400 ml of ethyl ether and washed three times with water. After evaporation and removal of excess ethyl 6-bromohexanoate by distillation under vacuum, 32.5 g of product F (yield: 100%) are obtained in the form of a brown oil. EXAMPLE 7

5- [(5-carbethoxypentyl) oxy] -2,3,3-trimethyl- (3H) -indole (product G)

A mixture of 23.1 g of product C, 32.4 g of ethyl 6-bromohexanoate and 20.1 g of potassium carbonate in 180 ml of acetone is refluxed for 8 hours and then cooled to room temperature. The reaction medium is filtered and evaporated under vacuum. The residue is dissolved in 600 ml of ethyl ether and washed three times with water. After evaporation and removal of the excess ethyl 6-bromohexanoate by distillation under vacuum, 41.8 g of product G (yield: 100%) is obtained in the form of a brown oil.

EXAMPLE 8

5- [(5-Carboxypentyl) oxy] -2,3,3-trimethyl-benz (e) indole (product H)

11.6 g of product F in 100 ml of 1M KOH are heated at 80 ^° C. for 30 minutes. After cooling to temperature ambient, the reaction medium is neutralized by addition of 1M hydrochloric acid. The precipitate formed is filtered and then washed with water and dried under vacuum in the presence of P ₂ O ₅ . 9 g of product H are obtained (yield: 84.2%).

EXAMPLE 9

5- [(5-carboxypentyl) oxy] -2,3,3-trimethyl- (3H) -indole (product I)

4.2 g of product G in 45 ml of 1M KOH are heated at 80 ^° C. for 30 minutes. After cooling to room temperature, the reaction medium is neutralized by adding 1M hydrochloric acid. The precipitate formed is filtered and then washed with water and dried under vacuum in the presence of P ₂ O ₅ . 2.5 g of product I are obtained (yield: 66.5%).

EXAMPLE 10

5- [(4-sulfobutyl) oxy] -2,3,3-trimethylbenz (e) indole, sodium salt (product J)

A mixture of 1.3 g of product E, 0.9 g of 1,4-butane sultone and 0.3 g of sodium hydroxide in 10 ml of ethanol is refluxed for two hours. After cooling to room temperature, the reaction medium is added to 100 ml of acetone with stirring. The precipitate formed is filtered and then washed with acetone and dried under vacuum. 1.8 g of very hygroscopic product J (Yield: 81.2%) are obtained.

EXAMPLE 11

2- [7- [8- (5-carboxypentyloxy) -1,3-dihydro-1,1-dimethyl-3- (4-sulfobutyl) -benz (e) indol-2-ylidene] -1,3,5 -heptatrienyl] -

1,1-dimethyl-3- (4-sulfobutyl) -1H-benz (e) indolium, inner salt, sodium salt (product K)

A mixture of 10.2 g of product H and 32.7 g of 1,4-butane sultone is heated at 115 ^° C. for 16 hours and then cooled. at room temperature. 120 ml of toluene are added, then the medium is filtered to recover the insoluble fraction. The precipitate is rinsed with acetone and dried under vacuum. 13.3 g of this solid is reacted with 15.2 g of 2- (6-acetanilido-1,3,5-hexatrienyl) -3,3-dimethyl-1- (4-sulfobutyl) benzene. indolium, internal salt, in 90 ml of ethanol. 2.9 g of triethylamine are gradually added and the mixture is refluxed for 5 minutes and then cooled to room temperature. 3.8 g of sodium acetate trihydrate are added and the mixture is stirred for 10 minutes. The precipitate formed is filtered and then washed with acetone and dried under vacuum. 18.5 g of product K are obtained (yield: 72.8%).

EXAMPLE 12

2- [5- [8- (5-carboxypentyloxy) -1,3-dihydro-1,1-dimethyl-3- (4-sulfobutyl) -benz (e) indol-2-ylidene] -1,3-pentadienyl ] -1,1-dimethyl-3- (4-sulfobutyl) -1H-benz (e) indolium internal salt, sodium salt (product L)

A mixture of 8.1 g of product H and 26.2 g of 1,4-butane sultone is heated at 120 ^° C. for 12 hours and then cooled to ambient temperature. 80 ml of toluene are added, then the medium is filtered to recover the insoluble fraction. The precipitate is rinsed with acetone and dried under vacuum. 10.6 g of this solid is reacted with

11.5 g of 2- (4-acetanilido-1,3-butadienyl) -3,3-dimethyl-1-

(4-sulfobutyl) -benz (e) indolium, internal salt, in 75 ml of ethanol. 2.2 g of triethylamine are gradually added and the mixture is refluxed for 5 minutes and then cooled to room temperature. 3 g of sodium acetate trihydrate are added and the mixture is stirred for 10 minutes. The precipitate formed is filtered and then washed with acetone and dried under vacuum. 15.2 g of product L (yield: 75.2%) are obtained.

EXAMPLE 13

2- [5- [6- (5-carboxypentyloxy) -1,3-dihydro-1,1-dimethyl-3- (4-sulfobutyl) -indol-2-ylidene] -1,3-pentadienyl] -1, 1-dimethyl-3- (4-sulfobutyl) -1H-benz (e) indolium, inner salt, sodium salt (product M)

A mixture of 10.8 g of product I and 40.8 g of 1,4-butane sultone is heated at 130 ° C for 8 hours and then cooled to room temperature. 80 ml of toluene are added, then the medium is filtered to recover the insoluble fraction. The precipitate is rinsed with acetone and dried under vacuum. 13.4 g of this solid is reacted with 16.3 g of the internal salt of 2- (4-acetanilido-1,3-butadienyl) -3,3-dimethyl-1- (4-sulfobutyl) -benz ( e) indolium in 175 ml ethanol. 3.2 g of triethylamine are gradually added and the mixture is refluxed for 5 minutes and then cooled to room temperature. 4.3 g of sodium acetate trihydrate are added and the mixture is stirred for 10 minutes. The precipitate formed is filtered and then washed with acetone and dried under vacuum. 18.2 g of product M (yield: 69.6%) are obtained.

EXAMPLE 14

2- [7- [8- (5-carboxypentyloxy) -1,3-dihydro-1,1-dimethyl-3- (4-sulfobutyl) -benz (e) indol-2-ylidene] -1,3,5 -heptatrienyl] -1,1-dimethyl-3- (4-sulfobutyl) -8- (4-sulfobutyloxy) -1H-benz (e) indolium, inner salt, sodium salt (product N)

A mixture of 0.6 g of product J and 1.7 g of 1,4-butane sultone is heated at 120 ^° C. for 16 hours and then cooled to ambient temperature. 5 ml of toluene are added, then the medium is filtered to recover the insoluble fraction. The precipitate is rinsed with acetone and dried under vacuum. 0.7 g of this solid is reacted with 0.9 g of 2- (6-acetanilido-1,3,5-hexatrienyl) -5- (5-carboxypentyloxy) -3,3-dimethyl-1- ( 4-sulfobutyl) -benz (e) indolium, inner salt, (obtained from the internal salt of 5- (5-carboxypentyloxy) -1- (4-sulfobutyl) -2,3,3-trimethylbenz (e) indolium, whose preparation is described in Examples 11 and 12, and dianilide hydrochloride glutaconic aldehyde) in 10 ml of ethanol. 0.14 g of triethylamine are gradually added and the mixture is refluxed for 5 minutes and then cooled to room temperature. 0.18 g of sodium acetate trihydrate is added and the mixture is stirred for 10 minutes. The precipitate formed is filtered and then washed with acetone and dried under vacuum. 1.1 g of product N (yield: 75.5%) are obtained. EXAMPLE 15

2- [7- [8- (4-sulfobutyloxy) -1,3-dihydro-1,1-dimethyl-3- (4-sulfobutyl) -benz (e) indol-2-ylidene] -1,3,5 -heptatrienyl] -1,1-dimethyl-3- (4-sulphobutyl) -8- (4-sulphobutyloxy) -1H-benz (e) indolium, inner salt, trisodium salt (product 0)

A mixture of 0.6 g of product J and 1.7 g of 1,4-butane sultone is heated at 120 ^° C. for 16 hours and then cooled to ambient temperature. 5 ml of toluene are added, then the medium is filtered to recover the insoluble fraction. The precipitate is rinsed with acetone and dried under vacuum. 0.7 g of this solid is reacted with 0.97 g of 2- (6-acetanilido-1,3,5-hexatrienyl) -3,3-dimethyl-1- (4-sulfobutyl) -5- ( 4-sulfobutyloxy) -benz (e) indolium, inner salt, sodium salt, (obtained from 1- (4-sulfobutyl) -5- (4-sulfobutyloxy) -2,3,3-trimethylbenzene) indolium, internal salt, sodium salt, the preparation is described previously, and dianilide hydrochloride of glutaconic aldehyde) in 10 ml of ethanol. 0.14 g of triethylamine are gradually added and the mixture is refluxed for 5 minutes and then cooled to room temperature. 0.18 g of sodium acetate trihydrate is added and the mixture is stirred for 10 minutes. The precipitate formed is filtered and then washed with acetone and dried under vacuum. 1.2 g of product O are obtained (yield: 79.1%). EXAMPLE 16

Sulfosuccinimidyl ester of product K (product P)

0.09 g of product K are solubilized in 5 ml of DMF. 0.11 g of sodium 1-hydroxy-3-succinimide-sulfonate and 25 mg of DiCyclohexylCarbodiimide are added. The reaction medium is stirred at room temperature for 24 hours and then filtered. 100 ml of ethyl ether are added to the filtrate. The precipitate formed is filtered and dried under vacuum. 105 mg of product P (yield: 95.4%) are obtained.

EXAMPLE 17

Succiniπiidyl ester of product K (product Q)

1.08 g of product K are solubilized in 50 ml of DMF. 0.69 g of N-hydroxysuccinimide and 0.31 g of DiCyclohexylcarbodiimide are added. The reaction medium is stirred at room temperature for 24 hours and then filtered. 250 ml of ethyl ether are added to the filtrate. The precipitate formed is filtered and dried under vacuum. 1.1 g of product Q (yield: 91.7%) are obtained.

EXAMPLE 18

p-nitrophenyl ester of product K (product R) 90 mg of product K and 17 mg of p-nitrophenol are solubilized in 2 ml of DMF. 25 mg of DiCyclohexylCarbodiimide are added. The reaction medium is stirred at room temperature for 24 hours and then filtered. 100 ml of ethyl ether are added to the filtrate. After filtration and drying under vacuum, 101 mg of product R (yield: 98%) are obtained. EXAMPLE 19

Succinimidyl ester of product L (product S)

0.24 g of product L and 0.12 g of TSTU (N, N, N ', N' - tetramethyl-O- (N-succinimidyl) uroniuna tetrafluoroborate) are solubilized in 5 ml of DMF. After stirring for 10 minutes, 64 mg of diisopropylethylamine are added. After stirring for 2 hours, 100 ml of ethyl ether are added. The precipitate formed is filtered off, washed with ethyl ether and dried under vacuum. 0.25 g of product S is obtained (yield: 92.6%).

EXAMPLE 20

Succinimidyl ester of product M (product T)

83 mg of product M and 40 mg of TSTU are solubilized in 5 ml of DMF. After stirring for 10 minutes, 25 mg of diisopropylethylamine are added. After stirring for 2 hours, 100 ml of ethyl ether are added. The precipitate formed is filtered, washed with ethyl ether and dried under vacuum. 90 mg of product T is obtained (Yield 96.8%).

EXAMPLE 21

Succinimidyl ester of product N (product U)

1.08 g of product N are solubilized in 50 ml of DMF. 0.57 g of N-hydroxysuccinimide and 0.26 g of DiCyclohexylCarbodiimide are added. The reaction medium is stirred at room temperature for 24 hours and then filtered. 250 ml of ethyl ether are added to the filtrate. The precipitate formed is filtered and dried under vacuum. 0.99 g of product U are obtained (yield: 84.2%).

EXAMPLE 22

Phenethyl amide of product L (product V)

93 mg of product S and 39 mg of phenethylamine are solubilized in 5 ml of DMF. After stirring for 45 minutes, 60 ml of ethyl ether are added. The precipitate formed is filtered off, washed with ethyl ether and dried under vacuum. 57 mg of product V are obtained (yield: 61.3%).

EXAMPLE 23

Protein labeling with S product

To a solution of 1 mg of protein (molecular weight 150 kDa) in 0.5 ml of phosphate buffer solution (0.1 M) is added 1 ml of carbonate-sodium bicarbonate buffer (0.1 M, pH 9.3 ) containing 10% of dimethylformamide, then 25 .mu.l of a solution of 9.1 mg of product S in 2 ml of dimethylformamide are added dropwise. The mixture is stirred for 30 minutes at room temperature. 200 μl of this mixture are eluted in fractions of 0.5 ml with phosphate buffer (0.1 M, pH 7.4, 10% dimethylformamide) on a Sephadex G25 / PD-10 column. (Amersham Biosciences) previously equilibrated with 25 ml of the same buffer. The dye coupled to the protein is separated from the unpulsed dye by gel permeation: the first color band (fractions 7 to 9) corresponds to the coupled dye and the second color band (fractions 14 to 17) corresponds to the free dye. Reading the optical densities at 685 nm indicates an average substitution rate of 4.7 dye molecules per molecule of antibody. The conjugate is stored at 4 ^° C., protected from light.

Claims

1. Marker capable of forming a covalent or non-covalent bond with a target molecule, consisting of a dye to which is covalently linked by one or more carbons of its chemical structure:

- one or more group(s) [FONC], and

- possibly one or more group(s) [SOL] said marker having the general formula:

[COLOR]

[COLOR] representing a dye chosen from the group comprising phthaleins, carbocyanines, merocyanines, porphyrins, phthalocyanines having one of the following structures (l) to (5):

(2)

(3)

(5)

in which :

Mi to M4, Mg, Mi ₂ , Qi to Q12, Di to Di ₆ , which are identical or different from each other, are chosen from the group comprising the following radicals or groups: hydrogen, hydroxyl, halogen, acetyl, amine, substituted amine, quaternary ammonium, phosphate, nitro, carboxylic acid and its salts, sulfonic acid and its salts, alkylcarboxy of 2 to 30 carbon atoms, alkyl, linear or branched, having 1 to 30 carbon atoms, cycloalkyl having 3 with 14 carbon atoms, alkyloxy having from 1 to 30 carbon atoms, haloalkyl having from 1 to 30 carbon atoms, hydroxyalkyl having from 1 to 30 carbon atoms, alkyl ester having from 2 to 40 carbon atoms, nitroalkyl having from 1 with 30 carbon atoms, carboxyalkyl having 2 to 30 carbon atoms, aminoalkyl having 1 to 30 carbon atoms, sulfoalkyl having 1 to 30 carbon atoms, aryl, aryloxy, arylalkyl, haloaryl, arylester, Q ₃ , Q ₆ , Q ₉ and Qi ₂ cannot represent aryl or aryloxy, on the condition that at least one of Qi to Qi ₂ represents H or an aromatic ring, on the condition that at least one of Di to Di ₆ represents H;

Zi and Z ₂ each independently represent the atoms necessary to complete an indole, benzindole or naphtindole nucleus; Z ₃ represents 0 or S; V and W are each independently chosen from CR ₇ R ₈ , 0, S, Se and NRg, where R ₇ , R ₈ and R ₉ are each independently chosen from hydrogen and a group (CH ₂ ) _m Rio, where m is an integer from 1 to 18 and Ri ₀ is selected from hydrogen, amine, substituted amine, quaternary ammonium, aldehyde, halogen, cyano, aryl, heteroaryl, hydroxyl, amide, acid sulphonic acid and its salts, carboxylic acid and its salts; n is an integer from 1 to 10, preferably from 1 to 7 and more preferably from 1 to 3; i is an integer from 1 to n;

R ₃ to R ₆ each represent, independently of each other, a hydrogen atom, an alkyl group, linear or branched in Ci-C ₃₀ , preferably in Ci-Ci ₈ , more preferably in C ₁ -C ₅ , cycloalkyl, aryl, aryloxy, nitroalkyl, alkylamine, substituted alkylamine, quaternary alkylammonium, alkylphosphate, alkylsulfonic acid and its salts;

Ti to T ₈ each independently represent a hydrogen atom, a halogen atom, a linear or branched Ci-C ₃₀ alkyl group, preferably Ci-Ci ₈ , more preferably Ci-C ₅ , sulfoalkyl, cycloalkyl, aryl, aryloxy, nitro, amine, substituted amine, quaternary ammonium, phosphate, sulfonic acid and its salts, ORn with R ₁₁ chosen from hydrogen and a C ₁ -C ₃₀ alkyl group, more preferably with Ru as defined previously; represent each independently of each other a hydrogen atom, a linear or branched alkyl group in preferably in more preferably cycloalkyl or aryl;

Bu, B ₂ i _/ B ₃ each independently represent a methine group (=CH-), mono or di-unsaturated cycloalkyl of 4 to 8 carbon atoms, aryl-mono or di-unsaturated cycloalkyl of 4 to 8 carbon atoms, or one of the following groups:

each of these groups being either unsubstituted or substituted by one or more of the following groups: linear or branched Ci-Ciβ alkyl, preferably Ci-C ₅ ;

Y represents a counterion chosen from the following ions: halide, p-toluenesulfonate, methanesulfonate, trifluoromethane-sulfonate, perchlorate, acetate, sodium, potassium, calcium, magnesium, lithium, ammonium and trialkylammonium; p is an integer from 0 to 8 necessary for the neutrality of the molecule; [FONC] each independently representing a group -X-A-Z, in which:

_- _{_} or branched, in C1-C30, preferably in Ci-Ci ₈ and more preferably in Ci-C _5;

- A is chosen from the group consisting of an alkylene group, or an alkylene-arylene group; - Z is a reactive chemical function chosen from the group comprising the functions carboxylic acid and its salts, sulfonic acid and its salts, acid anhydride, acid chloride, ester such as alkyl ester, p-nitrophenyl ester, succinimidyl ester, sulfosuccinimidyl ester, azido such as acyl azide, azidonitrophenyl, hydrazide, 3-acyl-l, 3-thiazolidine-2-thione, amine, substituted amine, quaternary ammonium, isocyanate, isothiocyanate, hydrazine, phthalimido, maleimide, haloacetamide, monochlorotriazine , dichlorotriazine, mono or dihalogenated pyridine, mono or dihalogenated diazine, aziridine, thiol, sulfonyl chloride, vinylsulfone, disulfide, methanethiosulfonate, hydroxyl, phosphoramidite, epoxy, aldehyde, carbonate, glyoxal, imidazolyl; [SOL] each independently representing a group -X'-A'-Z', in which:

- X' is chosen from the group consisting of an oxygen atom, a sulfur atom, an NR ₁ R ₂ group, Ri and R ₂ each being independently of the other a hydrogen atom or a group alkyl, linear or branched, in Ci-C ₃₀ , preferably in Ci-Ci ₈ and more preferably in Ci-C _5;

- A' is chosen from the group consisting of an alkylene group or alkylene-arylene group;

-Z' is either a polar group chosen from the group comprising carboxylic acid and its salts, sulfonic acid and its salts, amine, substituted amine, quaternary ammonium, carbohydrate, glycol, hydroxyl, nitro, phosphate, or a group nonpolar chosen from the group comprising linear or branched alkyl groups having from 1 to 30 carbon atoms, cycloalkyl having from 3 to 14 carbon atoms, alkyloxy having from 1 to 30 carbon atoms, haloalkyl having from 1 to 30 carbon atoms , hydroxyalkyl having from 1 to 30 carbon atoms, alkyl ester having from 2 to 40 carbon atoms, aryl, aryloxy, substituted arylalkyl, arylalkyl, haloaryl.

2. Marker according to claim 1, characterized in that the alkylene group is a hydrocarbon chain, cyclic, linear or branched, having two free bonds, containing from 1 to 30, preferably from 1 to 18 and even more preferably from 1 with 5 carbon atoms and which may in particular be a methylene, ethylene, propylene, 2-methylpropylene, n-butylene, i-pentylene, n-pentylene, hexylene chain and the arylene group is a aromatic group, having two free bonds, containing one or more aromatic rings, optionally substituted, including in particular a cyclopentadiene or phenylene group which may be substituted or unsubstituted.

3. Marker according to claim 1 or 2, characterized in that [FONC] is chosen from

-X- (CH ₂ ) _r -COOSu, -X- (CH ₂ ) _r -COOSuSO ₃ Na, -X- (CH ₂ ) _r -COOH, -X- (CH ₂ ) _r -SO ₃ H, -X - (CH ₂ ) _r -SO ₃ Na, -X- (CH ₂ ) _r -COO-C ₆ H ₄ -NO ₂ ,

-X- (C ₆ H ₄ ) - (CH ₂ ) _r -C00Su, -X- (CH ₂ ) _r -NHCOCH ₂ I, -X- (CH ₂ ) _r ~NCS, -X- (CH ₂ ) _r -C ₆ H ₄ CH (CH ₃ ) -COOSu, -N (CH ₃ ) ₂ - (CH ₂ ) _r -SO ₃ H, -X- (CH ₂ ) _r -0P [N (IPr) ₂ ] [CH ₂ CH ₂ CN],

4. Marker according to any one of claims 1 to 3, characterized in that [SOL] is chosen from -X- (CH ₂ ) _r -SO ₃ Na, -X- (CH ₂ ) _r -SO ₃ H , -X- (CH ₂ ) _r -C ₆ H ₃ (NO ₂ ) ₂ ,

-X- (CH ₂ ) _E -CH ₃ ,

5. Marker according to any one of claims 1 to 4, characterized in that it is fluorescent.

6. Marker according to any one of claims 1 to 5, characterized in that it is chosen from the group comprising the following structural molecules (6) to (10):

(6) in which have as defined previously; each of independently one of the other, can have the definition given previously for or may represent [GROUND] or [FONC], [GROUND] and [FONC] being as defined previously, and on the condition that at least one among represents [FUNC];

(7) in which are as defined previously, each independently of the other, can have the definition given previously for Ti to Ts, or can represent [SOL] or [FONC], [SOL] and [FONC] being such that defi ment, and on the condition that at least one among them , represents [FUNC];

(8) in which are as defined previously, each independently of the other, can have the definition given previously for Ti to T ₄ , or can represent [SOL] or [FONC], [SOL] and [FONC] being as defined previously, and on the condition that at least one among T ' ₁ to T ' ₄ represents [FONC];

(9) in which Q ₃ , Qç, Qg and Qi ₂ are as defined previously, each of independently of each other can have the definition given previously for or may represent [GROUND] or [FONC], [GROUND] and [FONC] being as defined previously, and on the condition that one at least Q' ₁ , Q' ₂ , Q' ₄ , Q' ₅ , Q' ₇ , Q' ₈ , Q' ₁₀ and Q' ₁ represents [FONC];

in which each of D'i to D'i ₆ , independently of each other, can have the definition given previously for Di to Oie, or can represent [FONC] or [SOL], [SOL] and [FONC ] being as defined previously, and on the condition that at least one among D'i to D ¹ _I6 represents [FONC].

7. Marker according to one of claims 1 to 6 characterized in that it is water-soluble, preferably at a concentration greater than 0.5%

(m/v), more preferably at a concentration greater than or equal to 2% (m/v), and even more preferably still greater than 10% (m/v).

8. Marker according to one of claims 1 to 6, characterized in that it is fat-soluble, preferably at a concentration greater than 0.5%

(m/v), more preferably at a concentration greater than or equal to 2% (m/v), and even more preferably even greater than 10% (m/v).

9. Marker according to any one of claims 1 to 8, characterized in that it is chosen from the group comprising

(1)

in which each of y, z, identical or different, is an integer equal to 1, 2, 3, 4, 5, 6, 7 or 8,

(2)

in which each of y, z and n, identical or different, is an integer equal to 1, 2, 3, 4, 5, 6, 7 or 8, Z represents -COOH or in which each of y, z and n, identical or different, is an integer equal to 1, 2, 3, 4, 5, 6, 7 or 8 Z represents -COOH or

Y represents SO ₃ ^~ or SO ₃ Na

particular

(3)

in which each of y, z and n, identical or different, is an integer equal to 1, 2, 3, 4, 5, 6, 7 or 8 Z represents -COOH or

Y represents SO ₃ ^" or SO ₃ Na in particular

(5)

Y represents SC > ₃ ^~ or SO ₃ Na in particular

in which each of y and z, identical or different, is an integer equal to 1, 2, 3, 4, 5, 6, 7 or 8 Z represents-COOH or

Y represents Sθ ₃ - or SO ₃ Na in particular

Y represents SO ₃ or SO ₃ Na particular

10. Process for preparing markers according to any one of claims 1 to 9, characterized in that it comprises a nucleophilic substitution reaction between: or else:

Z-A-L and [COLOR' ] - Nude

or :

[COLOR''] -L and Z-A-Nu

- Z and A being such as defined in claim 1;

- L representing a leaving group; _ Nu representing a nucleophilic group chosen from the group comprising -OH, -SH or -NRiR ₂ , Ri and R ₂ each being independently of the other a hydrogen atom or a linear or branched C alkyl group ₁ -C ₃₀ , preferably C ₁ -C ₁₈ ; more preferably in Ci-C ₅ ;

- [COLOR'] and [COLOR"] representing [COLOR] or a precursor or synthesis intermediate of [COLOR],

[COLOR] being as defined in claim 1.

11. Method according to claim 10, characterized in that the nucleophilic substitution reaction is a Williamson reaction in the presence of a base.

12. Method according to claim 10 or claim

11, characterized in that the leaving group L is chosen from the group comprising in particular a halogen or a methanesulfonate, para-toluenesulfonate group or a diazonium group.

13. Method according to any one of claims 10 to

12, characterized by the fact that [COLOR'] -Nu is obtained by the following successive reactions: 1. nitration of [COLOR'];

2. reduction of the product obtained in step 1;

3. optionally: either alkylation of the product obtained in step 2, or else diazotization of the product obtained in step 2 to obtain a diazonium salt then nucleophilic substitution on this diazonium function.

14. Method according to any one of claims 10 to

13, characterized by the fact that [COLOR' '] -L is obtained by the following successive reactions: 1'. nitration of [COLOR''];

2' . reduction of the product obtained in step l;

3' . diazotization of the product obtained in step 2' to obtain a diazonium salt;

4'. optionally: nucleophilic substitution on the diazonium function of the product obtained in step 3'.

15. Method according to any one of claims 10 to

14, characterized by the fact that it comprises a nucleophilic substitution reaction between: or else:

Z' -A' -L and [COLOR']- Nude

or :

[COLOR''] -L and Z '-A' -Nu - Z' and A' being as defined in claim 1;

- L, Nu, [COLOR'] and [COLOR"] being as defined previously in claim 15; - [COLOR' ]- Nu and [COLOR" ] -L being prepared according to the process of any one of claims 15 at 20.

16. Method according to any one of claims 10 to 15, characterized in that the nucleophilic substitution reaction is carried out by opening a cyclic molecule of formula:

17. Method for marking a target molecule with a marker according to any one of claims 1 to 9 or prepared according to any one of claims 10 to 16.

18. Formula Products

pY

in which Zi, V, p, Y are as defined previously,

R* ₃ represents an electronic doublet or represents

R ₃ as defined previously; μ is an integer equal to 0 or 1; R ₁₂ represents an alkyl group, linear or branched, saturated or unsaturated in C ₁ -C ₃₀ , preferably Ci-Ci _8/ more preferably in Ci-C ₅ , sulfoalkyl, cycloalkyl, aryl, aryloxy, preferably a methyl group, each of T" 1 to T' ₄ , independently of one another, can have the definition given previously for Ti to T ₄ or can represent [SOL] or [FONC], [SOL] and [FONC] being such as defined previously, and on the condition that at least one of T ' ₁ to T ' ₄ represents [FONC].

19. Products according to claim 18, of chemical formula:

Or

20. Use of a marker according to any one of claims 1 to 9 or prepared according to one of claims 10 to 16 in a method for marking biological molecules.

21. Use of a marker according to any of the claims 1 to 9 or prepared according to one of claims 10 to 16 in a coupling reaction in the presence of a peptide coupling agent, such as carbodiimide type reagents such as DCC (dicyclohexylcarbodiimide), carbonyldiimidazole, IDDQ ( l-isobutyloxycarbonyl-2-isobutyloxy-l, 2- dihydroquinoline), phosphonium type reagents such as BOP (benzotriazol-1- yloxytris- (dimethylamino) phosphonium hexafluorophosphate), uronium type reagents such as HBTu (o-benzotriazolyl- tetramethyluronium hexafluorophosphate) and TBTu (o-benzotriazolyltetramethyluronium tetrafluoroborate), Woodward reagents such as N-ethyl-5-phenylisoxazolium-3'-sulfonate, Curtius reagents (hydrazine and nitrite).

22. Use according to claim 20 or 21, characterized in that the coupling reaction is carried out in the presence of dimethylformamide.

23. Method for detecting a biological or non-biological molecule comprising the coupling of a marker according to one of claims 1 to 5 or prepared according to one of claims 10 to 16 with said molecule, and the detection of said molecule coupled to the marker by absorption spectrometry, fluorescence spectrometry, infrared spectrometry, electrophoresis, medical imaging in the near infrared or infrared.